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1.
Froehlich E Mandeville JS Weinert CM Kreplak L Tajmir-Riahi HA 《Biomacromolecules》2011,12(2):511-517
Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape. Among dendrimers, polyamidoamine (PAMAM) have received most attention as potential transfection agents for gene delivery, because these macromolecules bind DNA at physiological pH. The aim of this study was to examine the interaction of calf-thymus DNA with several dendrimers of different compositions, such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant DNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods, as well as atomic force microscopy (AFM), were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on DNA stability, aggregation, condensation, and conformation. Structural analysis showed a strong dendrimer-DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K(mPEG-G3) = 1.5 (±0.5) × 10(3) M(-1), K(mPEG-G4) = 3.4 (±0.80) × 10(3) M(-1), and K(PAMAM-G4) = 8.2 (±0.90) × 10(4) M(-1). The order of stability of polymer-DNA complexation is PAMAM-G4 > mPEG-G4 > mPEG-G3. Both hydrophilic and hydrophobic interactions were observed for dendrimer-DNA complexes. DNA remained in the B-family structure, while biopolymer particle formation and condensation occurred at high dendrimer concentrations. 相似文献
2.
S. G. Skuridin V. I. Popenko V. A. Dubinskaya V. A. Bykov Yu. M. Yevdokimov 《Molecular Biology》2009,43(3):453-464
The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated. 相似文献
3.
Milowska K Malachowska M Gabryelak T 《International journal of biological macromolecules》2011,48(5):742-746
α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers. 相似文献
4.
Milowska K Gabryelak T Bryszewska M Caminade AM Majoral JP 《International journal of biological macromolecules》2012,50(4):1138-1143
The aim of this work was to study the effect of phosphorus-containing dendrimers (generations G3 and G4) on the fibrillation of α-synuclein (ASN). The inhibition of fibril formation (filamentous and aggregates) is a potential therapeutic strategy for neurodegenerative disorders such as Parkinson's and other motor disorder neurodegenerative diseases. The interaction between phosphorus-containing dendrimers and ASN was studied by fluorescence spectroscopy. The decrease in the fluorescence intensity of intrisinic tyrosine was the most marked change in the fluorescence intensity observed upon addition of dendrimers. Furthermore, the effect of dendrimers on ASN fibril formation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by fluorescence assays using the dye thioflavin T (ThT). The results showed that phosphorus-containing dendrimers G3 and G4 inhibited fibril formation, when they were used in the ASN/dendrimer ratios 1:0.1 and 1:0.5. However, the higher concentrations of dendrimers did not show this effect. 相似文献
5.
Shcharbin D Janicka M Wasiak M Palecz B Przybyszewska M Zaborski M Bryszewska M 《Biochimica et biophysica acta》2007,1774(7):946-951
The detailed analysis of the interaction between PAMAM G4 dendrimer and serum albumins was performed using circular dichroism, isothermal titration calorimetry, capillary electrophoresis, zeta-potential and fluorescence polarization. It was shown that serum albumins and PAMAM G4 dendrimer form the complex with stoichiometry of 4-6:1 for G4:HSA and 4-5:1 for G4:BSA molar ratio. The possible sites of PAMAM G4 dendrimers binding to protein surface were discussed. Also, it has been proposed that dendrimer does not significantly affect the protein secondary structure studied by circular dichroism. 相似文献
6.
The effects of 4th and 5th generation cationic, neutral and anionic polyamidoamine (PAMAM) dendrimers on bilirubin absorbance and fluorescence were studied. Cationic and neutral PAMAM dendrimers shifted the bilirubin absorption maximum from 435 to 442-455 nm, increased the peak absorbance 1.5-fold, shifted the bilirubin fluorescence excitation and emission maxima, increased the fluorescence emission several-fold and significantly protected bilirubin against photodestruction. Using double fluorescence titration technique allowed to receive such constant of binding and the number of binding centers at 20 degrees C: for PAMAM G4 dendrimer, (2.4+/-1.4) x 10(6) (mol/l)(-1) and 0.07+/-0.012; for PAMAM G4-OH dendrimer, (3.1+/-1.3) x 10(6) (mol/l)(-1) and 0.08+/-0.014; for PAMAM G5 dendrimer, (7.6+/-3.6) x 10(6) (mol/l)(-1) and 0.09+/-0.02; and for PAMAM G5-OH dendrimer, (8.5+/-3.2) x 10(6) (mol/l)(-1) and 0.09+/-0.02. These effects can be explained by the formation of bilirubin-PAMAM dendrimer complexes and the formation of bilirubin monomers from tetramers. The formation of complexes sharply increased bilirubin solubility. We conclude that cationic and neutral PAMAM dendrimers bind bilirubin effectively and suggest that such dendrimers may serve as detoxication agents for hydrophobic endogenous toxins. 相似文献
7.
8.
Antos Shakhbazau Dzmitry Shcharbin Ihar Seviaryn Natalya Goncharova Svetlana Kosmacheva Mihail Potapnev Barbara Gabara Maxim Ionov Maria Bryszewska 《Molecular biology reports》2010,37(4):2003-2008
We report the use of polyamidoamine (PAMAM-NH2) dendrimers along with other non-viral vehicles for the in vitro transfection of human bone marrow mesenchymal stem cells
(hMSCs) and for engineering MSCs to secrete brain-derived neurotrophic factor (BDNF). Different generations of cationic polyamidoamine
dendrimers (generations 3–6) were tested on HEK 293T cells. hMSCs were then transfected with PAMAM-NH2 G4 dendrimers and Lipofectamine 2000, which elicited the expression of GFP reporter in around 6 and 20% of the cells, respectively.
Both vehicles were then shown to elicit the expression of BDNF in MSCs from a bicistronic cassette. Non-virally induced neurotrophin
expression may be a safe and easy method for adapting autologous stem cells for therapeutic treatment of diseases and neural
system injuries. 相似文献
9.
Steady state and dynamic fluorescence measurements have been used to investigate interaction between Bovine Serum Albumin (BSA) and fluorescence probe para-N,N-dimethylamino orthohydroxy benzaldehyde (PDOHBA), a structurally important molecule exhibiting excited state coupled proton transfer (PT) and charge transfer (CT) reaction. Fluorescence anisotropy, acrylamide quenching, and time resolved fluorescence measurements corroborate the binding nature of the probe with protein. The binding constant between BSA and PDOHBA has been determined by using Benesi-Hildebrand and Stern-Volmer equations. The negative value of ΔG indicates the spontaneity of this probe-protein complexation process. Observations from synchronous, three dimensional fluorescence spectra and circular dichroism spectra point toward the fact that the hydrophobicity as well as α-helix content of BSA are altered in presence of probe PDOHBA. The PT band of PDOHBA is found to be an excellent reporter for the mapping of destructive and protective behavior of SDS with variation of chaotrope concentration. 相似文献
10.
We report structural and dynamical aspects of DNAs from various sources including synthetic oligonucleotides in bulk buffer and as a complex with histone1 (H1). High-resolution transmission electron microscopic (HRTEM) studies reveal the structural change of the DNAs upon complexation with H1 leading to formation of compact-globular and hollow-toroidal particles. In order to explore the functionality of ligand binding of the DNAs and their complexes with H1, we have used two biologically common fluorescent probes Hoechst 33258 (H33258) and Ethidium (EB) as model ligands. Picosecond resolved fluorescence and polarization gated anisotropy studies examined that the minor groove binding of H33258 to the genomic DNA-H1 complex remains almost unaltered. However, the intercalative interaction of EB with the DNA in the complex is severely perturbed compared to that with the DNA in bulk buffer. Time-dependent solvochromic effect of the probe H33258 further elucidates the dynamical solvation, which is reflective of the overall environmental relaxation of the DNAs upon condensation by H1. We have also performed circular dichroism (CD) studies on the DNAs and their complexes with H1, which reveal the change in conformation of the DNAs in the complexes. Our studies in the ligand-binding mechanisms of the DNA-H1 complex are important to understand the mechanism of drug binding to linker DNA in condensed chromatin. 相似文献
11.
E Fredericq R Hacha P Colson C Houssier 《Journal of biomolecular structure & dynamics》1991,8(4):847-865
The condensation and the precipitation of rat liver chromatin upon addition of spermine4+, spermidine3+, hexamminecobalt(III)3+ and Mg2+ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg2+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg2+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates. 相似文献
12.
Yamini Thakur Mamta Tripathi Bharati Verma Rubi Khilari Rainy Agrawal Likheshwari 《Nucleosides, nucleotides & nucleic acids》2019,38(7):481-508
The polyadenylic acid [poly(A)] tail of mRNA plays a noteworthy role in the initiation of the translation, maturation, and stability of mRNA. It also significantly contributes to the production of alternate proteins in eukaryotic cells. Hence, it has recently been recognized as a prospective drug target. Binding affinity of bis(N-p-tolylbenzohydroxamato)Cobalt(II), [N-p-TBHA-Co(II)] (1) and bis(N-p-naphthylbenzohydroxamato)Copper(II), [N-p-NBHA-Cu(II)] (2) complexes with poly(A) have been investigated by biophysical techniques namely, absorption spectroscopy, fluorescence spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy, circular dichroism spectroscopy, viscometric measurements and through molecular docking studies. The intrinsic binding constants (Kb) of complexes were determined following the order of N-p-TBHA-Co(II)] > N-p-NBHA-Cu(II), along with hyperchromism and a bathochromic shift for both complexes. The fluorescence quenching method revealed an interaction between poly(A)-N-p-TBHA-Co(II)/poly(A)-N-p-NBHA-Cu(II). The mode of binding was also determined via the fluorescence ferrocyanide quenching method. The increase in the viscosity of poly(A) that occurred from increasing the concentration of the N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) complex was scrutinized. The characteristics of the interaction site of poly(A) with N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) were adenine and phosphate groups, as revealed by DRS-FTIR spectroscopy. Based on these observations, a partial intercalative mode of the binding of poly(A) has been proposed for both complexes. Circular dichroism confirmed the interaction of both the complexes with poly(A). The molecular docking results illustrated that complexes strongly interact with poly(A) via the relative binding energies of the docked structure as ?259.39eV and ?226.30eV for N-p-TBHA-Co(II) and N-p-NBHA-Cu(II) respectively. Moreover, the binding affinity of N-p-TBHA-Co(II) is higher in all aspects than N-p-NBHA-Cu(II) for poly(A). 相似文献
13.
The interaction mechanism between lipopeptide (daptomycin) and polyamidoamine (PAMAM) dendrimers
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Boontarika Chanvorachote Jiang Qiu Walaisiri Muangsiri Ubonthip Nimmannit Lee E. Kirsch 《Journal of peptide science》2015,21(4):312-319
The interaction mechanism of lipopeptide antibiotic daptomycin and polyamidoamine (PAMAM) dendrimers was studied using fluorescence spectroscopy. The fluorescence changes observed are associated with daptomycin–dendrimer interactions. The binding isotherms were constructed by plotting the fluorescence difference at 460 nm from kynurenine (Kyn‐13) of daptomycin in the presence and absence of dendrimer. A one‐site and two‐site binding model were quantitatively generated to estimate binding capacity and affinity constants from the isotherms. The shape of the binding isotherm and the dependence of the estimated capacity constants on dendrimer sizes and solvent pH values provide meaningful insight into the mechanism of interactions. A one‐site binding model adequately describes the binding isotherm obtained under a variety of experimental conditions with dendrimers of various sizes in the optimal binding pH region 3.5 to 4.5. Comparing the pH‐dependent binding capacity with the ionization profiles of daptomycin and dendrimer, the ionized aspartic acid residue (Asp‐9) of daptomycin primarily interact with PAMAM cationic surface amine. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
14.
Circular dichroism of bilirubin-amine association complexes: insights into bilirubin-albumin binding
Bichromophoric (4Z, 15Z)-bilirubin-IX alpha, the yellow-orange cytotoxic pigment of jaundice, adopts either of two intramolecularly hydrogen-bonded enantiomeric conformations that are in dynamic equilibrium in solution. The addition of optically active amines induces the pigment solutions to exhibit intense bisignate circular dichroism in the region of the bilirubin long wavelength uv-visible absorption band. The most intense circular dichroism Cotton effects, (delta epsilon) approximately equal to 130, are induced by beta-arylamines and are comparable to those exhibited by bilirubin complexes with serum albumin and other proteins. Like serum albumin and other proteins, the optically active base acts as a chiral complexation agent to induce an asymmetric transformation of bilirubin, whose induced bisignate circular dichroism Cotton effect is characteristic of exciton splitting of the component pyrromethenone chromophores. The amines thus serve as chiral templates for molecular recognition, and the complementary action of the amine complexation sites provides insight into the binding forces important in protein-bilirubin heteroassociation. 相似文献
15.
The complexation of (1→4) linked α-L-guluronate (G) and β-D-mannuronate (M) disaccharides with Mg(2+), Ca(2+), Sr(2+), Mn(2+), Co(2+), Cu(2+), and Zn(2+) cations have been studied with quantum chemical density functional theory (DFT)-based method. A large number of possible cation-diuronate complexes, with one and two GG or MM disaccharide units and with or without water molecules in the inner coordination shells have been considered. The computed bond distances, cation interaction energies, and molecular orbital composition analysis revealed that the complexation of the transition metal (TM) ions to the disaccharides occurs via the formation of strong coordination-covalent bonds. On the contrary, the alkaline earth cations form ionic bonds with the uronates. The unidentate binding is found to be the most favored one in the TM hydrated and water-free complexes. By removing water molecules, the bidentate chelating binding also occurs, although it is found to be energetically less favored by 1 to 1.5 eV than the unidentate one. A good correlation is obtained between the alginate affinity trend toward TM cations and the interaction energies of the TM cations in all studied complexes, which suggests that the alginate affinities are strongly related to the chemical interaction strength of TM cations-uronate complexes. The trend of the interaction energies of the alkaline earth cations in the ionic complexes is opposite to the alginate affinity order. The binding strength is thus not a limiting factor in the alginate gelation in the presence of alkaline earth cations at variance with the TM cations. 相似文献
16.
The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of ?181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes. 相似文献
17.
Two cobalt (II) complexes containing a dipyrido[3,2-a:2',3'-c]phenazine (dppz) base with the general formulation [Co(dppz)(dmp)(2)]Cl(2), where dmp is 4,7-dimethyl-1,10-phenanthroline ligand (4,7-dmp) (1) and 2,9-dimethyl-1,10-phenanthroline ligand (2,9-dmp) (2) were synthesized and characterized. Binding interactions of these complexes with calf thymus DNA were investigated by emission, absorption, circular dichroism, and viscosity studies, and the effects of the positions of methyl substitutions in phenanthroline coligands were investigated. The DNA binding constants obtained from the absorption spectral titrations decrease in the order of 1?>?2, which is consistent with the trend in apparent emission enhancement of the complexes on binding to calf thymus DNA. These observations were supported by circular dichroism spectroscopy and viscosity measurements and reveal that DNA binding affinity of the complexes depends on the position of methyl groups on the phenanthroline ligands. 相似文献
18.
Major attention has been focused on dendrimer-DNA complexes because of their applications in gene delivery systems. Dendrimers are also used to transport miRNA and siRNA in vitro. We examine the interaction of tRNA with several dendrimers of different compositions, mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) under physiological conditions using constant tRNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on RNA stability, aggregation, particle formation, and conformation. Structural analysis showed that dendrimer-tRNA complexation occurred via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K(mPEG-G3) = 7.6 (± 0.9) × 10(3) M(-1), K(mPEG-G4) = 1.5 (± 0.40) × 10(4) M(-1), and K(PAMAM-G4) = 5.3 (± 0.60) × 10(4) M(-1) show stronger polymer-RNA complexation by PAMAM-G4 than pegylated dendrimers. RNA remains in the A-family structure, whereas biopolymer aggregation and particle formation occurred at high polymer concentrations. 相似文献
19.
Intracellular degradation of genes, most notably within the endo-lysosomal compartment is considered a significant barrier to (non-viral) gene delivery in vivo. Previous reports based on in vitro studies claim that carriers possessing a mixture of primary, secondary and tertiary amines are able to buffer the acidic environment within the endosome, allowing for timely release of their contents, leading to higher transfection rates. In this report, we adopt an atomistic molecular dynamics (MD) simulation approach, comparing the complexation of 21-bp siRNA with low-generation polyamidoamine (PAMAM) dendrimers (G0 and G1) at both neutral and acidic pHs, the latter of which mimics the degradative environment within maturing 'late-endosomes'. Our simulations reveal that the time taken for the dendrimer-gene complex (dendriplex) to reach equilibrium is appreciably longer at low pH and this is accompanied by more compact packaging of the dendriplex, as compared to simulations performed at neutral pH. We also note larger absolute values of calculated binding free energies of the dendriplex at low pH, indicating a higher dendrimer-nucleic acid affinity in comparison with neutral pH. These novel simulations provide a more detailed understanding of low molecular-weight polymer-siRNA behavior, mimicking the endosomal environment and provide input of direct relevance to the "proton sponge theory", thereby advancing the rational design of non-viral gene delivery systems. 相似文献
20.
Combined multispectroscopic and molecular docking investigation on the interaction between delphinidin‐3‐O‐glucoside and bovine serum albumin
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Anthocyanin is one of the flavonoid phytopigments with specific health benefits. The interaction between delphinidin‐3‐O‐glucoside (D3G) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling. D3G effectively quenched the intrinsic fluorescence of BSA via static quenching. The number of binding sites and binding constant Ka were determined, and the hydrogen bonds and van der Waals forces played major roles in stabilizing the D3G–BSA complex. The distance r between donor and acceptor was obtained as 2.81 nm according to Förster's theory. In addition, the effects of pH and metal ions on the binding constants were discussed. The results studied by synchronous fluorescence, three‐dimensional fluorescence and circular dichroism experiments indicated that the secondary structures of the protein has been changed by the addition of D3G and the α‐helix content of BSA decreased (from 56.1% to 52.4%). Furthermore, the study of site marker competitive experiments and molecular modeling indicated that D3G could bind to site I of BSA, which was in the large hydrophobic cavity of subdomain IIA. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献